Immunohistochemical Analysis of Cellular Markers

For immunohistochemistry, the sections were incubated at 4°C overnight with 1∶100 dilutions of primary antibodies, including rabbit anti-active caspase 3 (BD Biosciences, San Jose, CA, USA), mouse anti-PCNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-endothelin 1 (GeneTex Inc., Hsinchu, Taiwan), rabbit anti-phospho-AKT (Ser473) (Cell Signaling Technology, Danvers, MA, USA), and anti-AKT (Cell Signaling Technology, Danvers, MA, USA). After washing with PBS, the sections were incubated with a 1∶100 dilution of the secondary antibody, either goat anti-rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or goat anti-mouse IgG (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), at room temperature. The protocol of immunohistochemistry was described previously [32] (link).